The ERBB2 (Her-2/neu) proto-oncogene encodes a member of a group of epithelial tyrosine kinase receptors involved in the initiation and progression of diverse malignancies including breast, ovarian, and gastric cancers (Engel and Kaklamani, Drugs 67, 1329-1341, 2007; Wong et al., Gynecol Obstet Invest 40, 209-212, 1995). ERBB2 gene amplification and overexpression leads to uncontrolled cell growth and survival, increased colony formation, (Bartsch et al., BioDrugs 21, 69-77, 2007) and impaired DNA repair (Pietras et al., Oncogene 9, 1829-1838, 1994). Several different immunotherapeutic approaches directed against ERBB2-expressing breast and ovarian tumors have been developed to date. Anti-ERBB2 antibody based immunotherapies, such as the monoclonal antibody trastuzumab, may be used to treat breast cancer patients with ERBB2 overexpression, but this approach has not been as efficacious in ovarian cancer patients (Bookman et al., official journal of the Am. Soc. Clin. Onc. 21, 283-290, 2003). Additionally, cancer vaccines have been used to induce specific anti-tumor immunity, but they produced only weak T-cell responses and did not induce objective tumor regression (Knutson et al., J Clin Oncol 23, 7536-7545, 2002; Peoples et al., J Clin Oncol 23, 7536-7545, 2005).
A T cell receptor is a complex of membrane proteins that participate in the activation of T cells in response to the presentation of antigen. Stimulation of the TCR is triggered by major histocompatibility complex molecules (MHC) on antigen presenting cells that present antigen peptides to the T cells and bind to the TCR complexes to induce a series of intracellular signaling cascades. The TCR is generally composed of six different membrane bound chains that form the TCR heterodimer responsible for ligand recognition. TCRs exist in alpha/beta and gamma/delta forms, which are structurally similar but have distinct anatomical locations and functions. In one embodiment, the TCR comprises a TCR alpha and beta chain, such as the nucleic encoding the TCR comprises a nucleic acid encoding a TCR alpha and a TCR beta chain. In another embodiment, an alpha or beta chain or both comprises at least one N-deglycosylation. Each chain is composed of two extracellular domains, a variable and constant domain. In one embodiment, the TCR comprises at least one murine constant region. The constant domain is proximal to the cell membrane, followed by a transmembrane domain and a short cytoplasmic tail. In one embodiment, the co-stimulatory signaling domain is a 4-1BB co-stimulatory signaling domain. The variable domain contributes to the determination of the particular antigen and MHC molecule to which the TCR has binding specificity. In turn, the specificity of a T cell for a unique antigen-MHC complex resides in the particular TCR expressed by the T cell. Each of the constant and variable domains may include an intra-chain disulfide bond. In one embodiment, TCR comprises at least one disulfide bond. The variable domains include the highly polymorphic loops analogous to the complementarity determining regions (CDRs) of antibodies. The diversity of TCR sequences is generated via somatic rearrangement of linked variable (V), diversity (D), joining (J), and constant genes. Functional alpha and gamma chain polypeptides are formed by rearranged V-J-C regions, whereas beta and delta chains consist of V-D-J-C regions. The extracellular constant domain includes a membrane proximal region and an immunoglobulin region.
TCR gene transfer has been developed over the last decade as a reliable method to generate large numbers of T-cells of a given antigen specificity for adoptive cellular therapy of viral infectious diseases, virus-associated malignancies, and cancer (Engels and Uckert, Mol Aspects Med 28, 115-142, 2007). The clinical feasibility of TCR gene therapy was first demonstrated in melanoma using a TCR specific for MART1, a commonly expressed melanoma antigen (Morgan et al., Science 314, 126-129, 2006). Adoptive transfer of MART1 TCR-transduced CD8+ T-cells used in fifteen patients resulted in durable engraftment of the transferred population and significant tumor regression in two patients, demonstrating a proof of concept of adoptive T-cell transfer (Morgan et al., Science 314, 126-129, 2006). A higher affinity MART-1-specific TCR that conferred improved functional avidity and clinical efficacy in melanoma was later identified, although with greater incidence of vitiligo, uveitis and hearing loss resulting from collateral destruction of normal melanocytes (Johnson et al., Immunol 177, 6548-6559, 2006; Johnson et al., J Blood 114, 535-546, 2009).
ERBB2-directed TCR gene therapy would appear to hold significant promise for common epithelial cancers. However, isolation of highly avid ERBB2-specific TCRs directly from cancer patients has been challenging and has not been tested clinically. One promising strategy to generate ERBB2-specific T-cells relies on vaccination of patients bearing ERBB2+ tumors with powerful immune regimens that can overcome immunological ERBB2 self-tolerance and prime preexisting T-cell immunity. Administration of an autologous, matured dendritic cell (DC) vaccine pulsed with ERBB2-derived HLA class I and II peptides to HLA-A2+ patients with ERBB2+ breast tumors was shown to efficiently prime ERBB2-specific T-cells, increase their frequency, and result in tumor regression in some patients in an ERBB2/DC vaccine study (Czerniecki et al., Cancer Res 67, 1842-1852, 2007). Although cytotoxic T-lymphocytes (CTLs) specific for various immunogenic ERBB2 peptides have been described, they often exhibit both poor functional avidity and tumor reactivity.
Therefore there is a need in the art for optimizing T cell based adoptive immunotherapy and for generating potent CD8+ T-cells highly specific for an ERBB2 epitope demonstrating high functional avidity and tumor reactivity against tumor cells expressing endogenous antigen. This invention addresses this need.